Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

Epstein-Barr virus (EBV) infection in infancy.

BACKGROUND: Epstein-Barr virus (EBV) has been shown to be the cause of infectious mononucleosis (IM) and has more complicated associations with several malignant diseases. These EBV associated diseases provide a strong incentive for the development of an EBV vaccine. Most primary EBV infection during infancy and early childhood is mild or subclinical. Little is known about its infection in infancy. The pattern of EBV serological response during infancy may be important for vaccine management. OBJECTIVES: this study has served to clarify the epidemiology and serology of primary EBV infection during early infancy. STUDY DESIGN: longitudinal serum samples from 66 Hong Kong infants were tested for EBV antibodies by immunofluorescence. Cord blood and sequential serum samples from these infants were taken at birth and then at 4-month intervals up to 2 years of age. RESULTS: maternal antibodies were present at different levels in all cord blood specimens and in serum samples of 8 infants at 4-month of age. Evidenced by VCA-IgG seroconversion, 60.6% (40/66) infants were infected during the first 2 years of life. One episode occurred before 8 months of age but, thereafter and for the remaining 16 months of follow-up until the infants were 2 years of age, the infection occurred at essentially a constant rate affecting about 20% of the remaining seronegative infants every 4 months. CONCLUSIONS: the abrupt onset of the infection after a delay of 8 months is a remarkable feature of primary EBV infection during infancy, which implicates a protective role for maternal antibodies. Persisting maternal antibodies may additionally serve to contain the infection once it occurred. This may partly explain why, unlike during adolescence, primary EBV infection early in life is usually asymptomatic.     

A reporter system for Epstein-Barr virus (EBV) lytic replication: anti-EBV activity of the broad anti-herpesviral drug artesunate

Epstein-Barr virus (EBV) is associated with severe human diseases. Therapies with conventional anti-herpesviral drugs are mostly ineffective so that novel drugs are urgently needed. As cell culture-based evaluation systems are required, a GFP (green fluorescent protein) reporter system was generated, which was conceived for an easy quantitation of lytic EBV replication and the analysis of EBV drug sensitivity. A reporter construct was generated on the basis of an EBV plasmid mini-replicon which enabled an episomal maintenance and selection of stably transfected Raji and 293T cell clones. Controlled by the viral lytic origin of DNA replication (oriLyt), this reporter construct could be activated through experimental EBV infection or through chemically stimulated reactivation from EBV latency. Using this system, the sensitivity of EBV to the broad-spectrum anti-herpesviral drug artesunate could be demonstrated: (i) artesunate inhibits EBV in the low micromolar range, (ii) two different strains of EBV are equally artesunate-sensitive, (iii) inhibition is detectable similarly in EBV-infected epithelial cells or lymphocytes, and (iv) the mode of antiviral action is based on a block of viral immediate early protein synthesis. The data demonstrate the usefulness of this reporter system for the quantitation of EBV replication and for determining EBV drug sensitivity.     

Stimulation of antibodies to Epstein-Barr virus (EBV) in acute viral infections

Summary Acute and convalescent sera from 77 patients with serologically confirmed influenza, measles and adenovirus infections and from 36 healthy controls were tested for the level of antibodies to Epstein-Barr (EB) virus. In the three groups of patients significantly higher titers of antibodies to EB viral capsid antigen (VCA) were found as compared to the controls. In 19 patients twofold or higher rise in antibody titers between the first and second blood sample was demonstrated.It is suggested that in patients with influenza, measles or adenovirus infections, involvement of lymphocytes leads to reactivation of EB virus and antibody formation is stimulated.     

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the parotid gland

A case of undifferentiated carcinoma of the salivary gland occurring in the parotid gland of a southern Chinese was reported. Tumour cells showed immunofluorescence for Epstein-Barr virus (EBV)-associated nuclear antigen, and DNA hybridization demonstrated the presence of EBV-DNA in tumour tissue. The findings in this case, together with previous reports, suggest a causal relationship between EBV and salivary gland carcinoma. The relationships between EBV and undifferentiated epithelial tumours of the salivary glands, nasopharynx and thymus are also discussed.     

HLA antigens in adults negative for antibody to Epstein-Barr virus (EBV)

Evidence for a genetic component in susceptibility to Epstein-Barr virus (EBV) infection was sought by comparing HLA A and B phenotypes of EBV antibody-negative Scottish medical students and randomly chosen antibody-positive controls. No statistically significant differences were observed, but three antigens, (A10, A29 and B15), were relatively underrepresented in the EBV seronegative group; findings which agree with data previously reported from a similar study in Los Angeles. A strong association between the HLA A1/Blank phenotype and EBV seronegativity, evident in the Los Angeles population, was not confirmed in the present study.     

Epstein-Barr virus (EBV)--lymphoid cell interactions. II. The influence of the EBV replication cycle on natural killing and antibody-dependent cellular cytotoxicity against EBV-infected cells.

We investigated the influence of the Epstein-Barr virus (EBV) replication cycle on natural killing (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) against EBV-infected cells. Peripheral blood lymphocytes from healthy EBV-seropositive and -seronegative donors were separated on Ficoll-Hypaque gradients and used as effector cells in the standard 51Cr release assay to measure NK and ADCC. EBV-genome positive RAJI and DAUDI cells superinfected with either the non-transforming P3HR-1 EBV or the transforming B95-8 EBV were used as targets. The results obtained show that most normal individuals have ADCC and NK activity against P3HR-1 EBV-infected RAJI cells. Both the cytotoxic activities increased with the proportional increase in effector/target (E/T) ratios, assay incubation time, dose of the infecting virus and the time of pre-infection with EBV. Moreover, the data obtained indicate that different immune mechanisms are effective at different stages of the virus replication cycle. During the early stages of virus replication, EBV-superinfected cells are more susceptible to ADCC than to NK, whereas in later stages the susceptibility to NK is increased significantly and appears to play a more dominant role. The nature of the target cells or the strain of EBV used to superinfect these targets did not influence their susceptibility to ADCC and NK activity; however some quantitative differences were found. Using metabolic inhibitors such as cytosine arabinoside, phosphonoacetic acid, actinomycin D, cycloheximide and puromycin, it was found that new DNA synthesis is not essential but some RNA and protein synthesis is necessary, late in the viral cycle, for the superinfected cells to become susceptible to NK and ADCC.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.     

Epstein-Barr virus (EBV) and gastric carcinoma in Malaysian patients

Epstein-Barr virus (EBV) has consistently been detected in the tumour cells of nasopharyngeal carcinoma and lymphoepithelial-like carcinoma of the salivary glands, and have occasionally been found in similar tumours at other sites. Moreover, recent studies from various parts of the world including the Orient have shown about 10% of gastric carcinomas to be EBV-associated. We studied 50 gastric carcinomas from Malaysia to investigate its association with EBV in the Malaysian population. They comprised 37 intestinal and 13 diffuse type carcinomas from 32 male and 18 female patients, age range from 29 to 86 years with an ethnic distribution of Malay: Chinese: Indian with the ratio of 4: 27: 19. EBV gene and gene-expression were examined in sections of formalin-fixed, paraffin-embedded tissue using commercially available probes for detecting EBV encoded RNAs (EBERs) by in situ hybridization and monoclonal antibodies to EBV latent membrane protein-1 (LMP-1) by standard immunohistochemistry. Five of 50 gastric carcinomas showed EBER intranuclear positivity in all tumour cells but no cases expressed LMP-1. The EBV-associated cases were classified as intestinal type in 4 and diffuse type in one case and all were histologically unremarkable. EBV-positive tumours were found in 3 Chinese and 2 Indian patients with none in the small Malay group. Four EBV-positive tumours were in male patients, with age-range of 65 to 86 years. We conclude that our findings of about 10% of Malaysian gastric carcinomas being EBV-associated is in line with the results from other parts of the world and from other ethnic groups.     

Transfection of human lymphocytes with cloned Epstein-Barr virus (EBV) DNA

Human primary cord-blood lymphocytes were transfected, using the DEAE-dextran technique, with a set of seven largely overlapping clones jointly covering the whole M-ABA Epstein-Barr virus (EBV) genome. Three fragments, cosmids cMB-14 and cM301-99 and plasmid pM966-20, were able to stimulate transient cellular DNA synthesis, blastic transformation, and clumps formation, as well as to prolong the life span from a maximum of 2 weeks in control cultures to up to 6 weeks. The fragments stimulating DNA synthesis also expressed this property in mutual combinations or when combined with cosmid cMSal-A or cM302-21. Their use with any other fragments in cotransfection did not result in further DNA synthesis stimulation. Cosmids cM302-23 and cMSal-B suppressed this effect. Cosmid cM301-99 but not cM302-23 induced transient EBNA-1 formation in about 1% of lymphocytes. Lymphocytes transfected with single fragments or their combinations failed to grow into immortalized cell lines. The results suggest that transient expression of viral functions at levels achievable by transfection is not sufficient for cell immortalization.     

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups. © 1996 Wiley-Liss, Inc.     

Infection of the human T-cell-derived leukemia line Molt-4 by Epstein-Barr virus (EBV): induction of EBV-determined antigens and virus reproduction

We have reported previously that human T-cell-derived Molt-4 cells become susceptible to Epstein-Barr virus (EBV) infection after implantation of functional EBV receptors into the plasma membranes of Molt-4 cells (Volsky et al., 1980). In the present work, we expand this finding by analyzing the following: (i) Virus adsorption vs viral penetration—using [³H]thymidine-labeled EBV, we demonstrate that the virus could adsorb to both the untreated and the receptor-implanted Molt-4 cells. However, only the altered cells became susceptible to EBV penetration followed by the viral antigen synthesis; (ii) EBV substrain specificity of infection—EBV P3HR-1 virus induced the nuclear (EBNA), early (EA), and virus capsid (VCA) antigens, whereas EBV B-95-8 virus induced only EBNA; (iii) virus reproduction—in situ hybridization was used to demonstrate the EBV-DNA synthesis in the P3HR-1 virus-infected cells. In addition, immature herpes-like particles were observed in electron micrographs of infected cells. It is concluded that EBV infection of human T-cell-derived Molt-4 cells may lead to a full viral-lytic cycle in a portion of infected cells. The results suggest, however, that the primary EBV productive infection may not necessarily involve any immunofluorescence-detectable EBNA synthesis.     

Vero cell-expressed Epstein-Barr virus (EBV) gp350/220 protects marmosets from EBV challenge

The Epstein-Barr virus (EBV) major membrane antigen, gp350/220, was purified from expressing, genetically engineered Vero cells. The antigen, formulated either with alum or Freund's adjuvant, was inoculated into EBV infection-susceptible marmosets. After several injections, most of the marmosets developed anti-gp350/220 antibodies, and several exhibited virus-neutralizing activity. The immune response elicited by the alum-absorbed antigen proved to be protective upon virus challenge of the inoculated animals. Protection did not correlate with the presence of neutralizing antibodies.     

Epstein-Barr virus (EBV) DNA levels in palatine tonsils and autologous serum from EBV carriers

A real-time polymerase chain reaction was employed to detect and quantitate Epstein-Barr virus (EBV) DNA in tonsils and autologous sera from EBV-seropositive children. EBV DNA was found in 95% of tonsils from 21 children and in 50% from 18 children with serum IgG titers to the virus capsid antigen (VCA) of > or =1:160 and 1:10 to 1:80, respectively (P = 0.002). Tonsils from children with titers > or =1:160 harbored more EBV DNA copies per mg tissue (mean, 1,237; range, < 2-13,998) than from children with titers 1:10 to 1:80 (mean, 23; range, < 2-226; P < 0.0001). By contrast, EBV DNA was detected only in serum from 25% of 20 children with titers > or = 1:160. Thus, ample differences in tonsillar EBV replication are mirrored inconstantly by detectable EBV in autologous serum suggesting that EBV DNA quantitation in tonsils may serve for refined monitoring of individuals at risk of EBV-associated lymphoproliferation.